Programmable Ligand Detection System in Plants through a Synthetic Signal Transduction Pathway

Abstract

BackgroundThere is an unmet need to monitor human and natural environments for substances that are intentionally or unintentionally introduced. A long-sought goal is to adapt plants to sense and respond to specific substances for use as environmental monitors. Computationally re-designed periplasmic binding proteins (PBPs) provide a means to design highly sensitive and specific ligand sensing capabilities in receptors. Input from these proteins can be linked to gene expression through histidine kinase (HK) mediated signaling. Components of HK signaling systems are evolutionarily conserved between bacteria and plants. We previously reported that in response to cytokinin-mediated HK activation in plants, the bacterial response regulator PhoB translocates to the nucleus and activates transcription. Also, we previously described a plant visual response system, the de-greening circuit, a threshold sensitive reporter system that produces a visual response which is remotely detectable and quantifiable.Methodology/Principal FindingsWe describe assembly and function of a complete synthetic signal transduction pathway in plants that links input from computationally re-designed PBPs to a visual response. To sense extracellular ligands, we targeted the computational re-designed PBPs to the apoplast. PBPs bind the ligand and develop affinity for the extracellular domain of a chemotactic protein, Trg. We experimentally developed Trg fusions proteins, which bind the ligand-PBP complex, and activate intracellular PhoR, the HK cognate of PhoB. We then adapted Trg-PhoR fusions for function in plants showing that in the presence of an external ligand PhoB translocates to the nucleus and activates transcription. We linked this input to the de-greening circuit creating a detector plant.Conclusions/SignificanceOur system is modular and PBPs can theoretically be designed to bind most small molecules. Hence our system, with improvements, may allow plants to serve as a simple and inexpensive means to monitor human surroundings for substances such as pollutants, explosives, or chemical agents.

GoldenBraid 2.0: A Comprehensive DNA Assembly Framework for Plant Synthetic Biology

“…Engineering enhanced abiotic stress tolerance for growth in marginal lands, turning C3 plants into C4 plants (Caemmerer et al, 2012), constructing whole-organism biosensors or sentinels (Antunes et al, 2011), engineering highly challenging metabolic routes (Farre et al, 2012), and combinations of these are just some examples of high-impact goals within the reach of biotechnologists. Also, it has not escaped our notice that the proposed grammar can be easily adopted by other nonplant systems as well.…”

Transplantation of prokaryotic two-component signaling pathways into mammalian cells

“…Each gene coding for a TCS protein was codon-optimized for expression in human cells and placed under the control of a constitutive CMV immediate early promoter. The amino acid sequence of each HK and RR was preserved, whereas the RR sequences were augmented with three minimal VP16 transactivating domains at the C terminus (31). Nuclear localization signal (NLS) was not fused to RRs as we reasoned that NLS would preclude RR from interacting with nonnuclear HK.…”

“…Small size and modularity of these pathways have already made them a target for rewiring and synthetic application in prokaryotic cells (28)(29)(30) as well as Arabidopsis (31). There was also a report on functional complementation by a plant-derived HK in yeast (32).…”

Effect of Dimethoate on Object Recognition Memory in Wistar Rats and Essay of Treatment with Nettle

“…This process therefore makes it possible to integrate and reorganize an already formed memory. By this means, in rodents, when a stimulus is previously experienced and reactivated, the hippocampus permits discrimination between the familiar and the new with a spontaneous and natural tendency to interact more with novelty [27]. Dysfunction in the OP induced cholinergic system in the hippocampus and/or perirhinal cortex may be suggested as a cause of alteration of short-term recognition memory.…”